We propose to develop a novel technology that allows one to image the spatiotemporal dynamics of the epigenetic functional status of histones within the chromatin in real time, thus enabling 4D-nucleome imaging in living cells at single cell level. The proposed molecular imaging system is developed by using a one-bead-one-compound (OBOC) combinatorial library screen to identify short peptide(s) that activate fluorescence of organic dyes or molecular rotors. Activation of fluorescence is coupled to alterations of their chemical environment including conformational change upon ligand binding and phosphorylation, acetylation, methylation, or ubiquitination of the peptide. These peptides can then be genetically fused to target proteins such as histones to enable functional cellular imaging in living cells in real time. We will adapt our newly developed technology to the epigenetics studies in this application. Hypothesis: The Genetically Encoded Small Illuminant (GESI) technology, comprised of OBOC combinatorial peptide library design and serial screening of huge arrays of immobilized bead (~1 million diversities) under pre-defined conditions, enables identification of short peptide- dye pairs that can be used as genetically encoded illuminants to probe post-translational modification of histones in nucleosomes, temporary and spatially in living cells in real time, thus enabling 4D nucleome imaging. Fluorescently activated GESI sites can be covalently marked for subsequent correlative fluorescent and electron microscopy, and chromatin precipitation via dye/GESI interaction. Impact: GESI peptides can specifically bind to and activate the fluorescence of selected organic dyes. Some GESI peptides will do so only after binding to cellular components such as Ca2+, conformational changes or post-translational modifications (PTMs). Therefore, when expressed in a living cell, they can illuminate the spatiotemporal regulation and modification of proteins of interest. The genetic illuminants are small (1200-1900 daltons), thus can be readily inserted along the sequence of the native proteins without interfering with their physiological functions. Multiplexing is possible and allows us to study cross talks of different histone PMTs and/or recruitments of histone binding proteins in real time. Specific aims of the proposed project are: Aim 1. To design and synthesize a series of organic dyes suitable for GESI reporting in the nucleus of living cells. Aim 2. To develop GESIs to track the spatiotemporal dynamics of subtype of histone H2A (H2AX and H2AZ). Aim 3. To develop GESIs to newly identify acylations status of N-terminus histone H2A/H3 inside living cells.